Heart failure (HF) is a significant health problem in the elderly population, including in Veterans. The basis for the diminished function of the old heart (OH, 19-22 month) and increased propensity to develop HF is only incompletely explored and poorly treated. Preliminary results show that in OH cardiac function is diminished, cardiac myocytes (CM) contract poorly, and decreased Mitochondrial (Mito) respiratory function occurs. Our preliminary results also show novel findings that in CM from OH versus young heart (YH, 2-3 month) the Mitochondrial Calcium Uniporter (MCU) Complex (MCUC) has markedly decreased calcium (Ca2+) conductance resulting in decreased Mito matrix freeCa2+ concentration ([Ca2+]m) which leads to decreased Mito respiratory, cardiac metabolic, and contractile function. Specific MCUC member proteins show decreased levels in OH CM. The level of the Essential MCU Regulator (EMRE) is decreased by 70% in OH CM. In the absence of EMRE, MCUC Ca2+ conductance is lost. In addition MCU shows a more modest 30% decline in OH CM. The principle hypothesis is that the performance of the OH can be markedly improved by restoring EMRE and MCU levels in OH CM (OH+EMRE+MCU) using adeno-associated viral vector (AAV)-based expression of transgenes (tges) encoding EMRE and MCU or pharmacological inhibition of Mito Ca2+ export. To test this hypothesis we pursue three closely linked Aims. In Aim 1 we enhance MCUC Ca2+ conductance by restoring EMRE and MCU in OH towards the YH range and determine its influence on Mito and cytosolic Ca2+ handling, Mito respiratory, cardiac metabolic and contractile function, and animal survival. We also inhibit the Mito sodium- Ca2+ exchanger (mNCLX) and Mito Ca2+ export with a pharmacological compound, returning [Ca2+]m to the YH level and improving CM contraction. In Aim 2 we determine if maladaptive consequences occur in OH+EMRE+MCU, especially increased CM death and increased myocardial infarct size (MI) with ischemia/reperfusion (I/R). Preliminary results show a decrease in MI size in OH+EMRE+MCU versus (vs) OH. Influences of dominant negative (dn) dnEMRE and dnMCU expression on Mito Ca2+ handling and CM contraction in YH and OH are investigated. We compare rescue effects in OH+EMRE+MCU vs expression of a SERCa2 tge in OH (OH+SERCa2). Preliminary results show similar basal and maximal ex vivo cardiac function in OH+EMRE+MCU and OH+SERCa2. In Aim 3 we explore molecular mechanisms mediating the marked decrease in EMRE protein levels in OH CM. Preliminary results point to an interaction of miRNA 215, which is 3.4-fold increased in OH CM, with the 5' UTR of EMRE mRNA inhibiting translation initiation. We also explore if chromatin remodeling to a more open state occurs in OH+EMRE+MCU vs OH with increased DNA accessibility enabling increased gene transcription, including of the SERCa2 gene. Chromatin remodeling involves chromatin remodeling motors which require ATP hydrolysis. Studies in YH, OH, and OH+EMRE+MCU are conducted with collaborators at San Diego VA and UCSD who have the expertise and equipment to perform the following experiments. We use the Assay for Transposase-Accessible Chromatin with deep Sequencing (ATAC-Seq) and Chromatin Immunoprecipitation Sequencing (Chip-Seq) with a Polymerase II antibody to probe DNA accessibility for gene transcription. Chip-Seq pathway enrichment and RNA Sequencing (RNA-Seq) are used to determine changes in gene expression. To establish a link to increased ATP levels we use CM from OH+EMRE+MCU and OH with or without the protonophore FCCP which rapidly dissipates the proton gradient and inhibits ATP formation. Limited preliminary results, which need to be confirmed and extended, from ATAC-Seq and Chip-Seq indicate increased DNA accessibility in OH+EMRE+MCU vs OH CM. Chip-Seq pathway enrichment analysis and RNA-Seq data indicate a shift in the gene expression profile to mRNAs encoding proteins linked to cardiac muscle contraction and muscle structure development in OH+EMRE+MCU vs OH. The closely linked Aims will lead to new knowledge and may result in novel therapeutic approaches.